Production, Purification and Characterization of the Hen Egg‐White Lysozyme Inhibitor from Enterobacter cloacae M‐1002

Three hen egg‐white lysozyme inhibitor producing strains, Enterobacter cloacae M‐1002, E. sakazakii M‐1204, and Erwinia rhapontici H‐55, were isolated from the soils of Taiwan. E. cloacae M‐1002 appeared to be a promising inhibitor producing strain. One inhibitor was isolated from the culture broth of this strain. Maximum lysozyme inhibitory activity was obtained when the bacterium was grown aerobically in a medium consisting of 0.75% glucose, 0.25% beef extract, 1.0% polypeptone, and 0.25% sodium L‐glutamate (pH 70) at 37 °C after 36–48 hrs. A hen egg‐white lysozyme inhibitor was isolated from the culture broth of this strain. The inhibitor was purified from the culture supernatant of E. cloacae M‐1002 by ammonium sulfate fractionation, DEAE‐Sepharose CL‐6B column chromatography and Fractogel TSK HW‐55 (S) gel chromatography. Molecular weight of the purified lysozyme inhibitor was estimated to be 18, 000–20, 000 by SDS‐PAGE and HPLC, and was composed of 71% amino acid and 23% total sugar. Serine, glycine, and alanine in a 3:2:1 molar ratio were the major amino acids, calculated to be 32.8, 20.3, and 11.4% (mol%), respectively. Glucose and mannose were the major sugar components of the inhibitor. The inhibitor was stable at pH 5 to 8 and was stable under 50 °C. Only hen egg‐white lysozyme was inhibited by the purified inhibitor but not the other tested enzymes such as lysozyme of celery, turnip; lytic enzyme of Pseudomonas aeruginosa M‐1001; chitinase/lysozyme of P. aeruginosa K‐187; or cellulase and xylanase of Streptomyces actuosus A‐151 and Aspergillus sp. G‐393. The inhibition of lysozyme to the bacterial cell lytic activity by the purified inhibitor was 100%.